The lysis/resealing technique is described in patents EP-A-101 341 and EP-A-679 101. The latter describes a relatively complex installation which comprises, firstly, a refrigerated housing for the lysis portion, in which there is placed an assembly comprising single-use elements, including a dialysis cartridge, tubes, chicane type or serpentine pouches and removable metal elements, such as a serpentine cooling arrangement, and, secondly, a housing for the resealing, which housing is provided with reheating means and in which a single-use assembly of plastics material is placed.
The red corpuscles, which have been separated from the plasma beforehand and which are subjected to weak ion forces (in a hypotonic medium), swell until they reach a critical volume, at which the membrane is distended to the point of becoming permeable to ions and macromolecules. Examination under a microscope of the erythrocyte membranes then reveals the appearance of pores which measure from 20 to 500 nm, as a result of which haemoglobin may escape (P. Seeman J. Cell. Biol. 1967, 32(1): 55-70). Restoration of the isotonicity of the suspension medium brings about closure of the pores, making the membrane impermeable to macromolecules. Only permeability to ions is maintained.
The hypotonic shock is brought about by causing the red corpuscles to circulate in the «blood» compartment of a dialyser, preferably having hollow fibres, and causing a hypotonic solution to circulate in counter-current in the «dialysate» compartment. The advantage of this technique resides in the confinement of the red corpuscles during the hypotonic shock, which allows losses of constituents which are essential to the life of those cells to be considerably reduced. Thus, the half-life of the red corpuscles is not significantly modified in vivo.
The advantage of using red corpuscles as vehicles for medicaments, in comparison with other techniques, such as encapsulation in liposomes or microspheres, resides substantially in that those corpuscles have «natural» biocompatibility, are completely biodegradable according to a well-known process, have a relatively long in vivo life expectancy (approximately 120 days) and in that various chemical and therapeutic molecules can be encapsulated therein.
The process of internalisation by lysis and resealing of the erythrocytes is a complex multi-factor phenomenon. Some significant physical/chemical parameters which have a bearing on the variability of the results are the concentration in terms of haemoglobin before dialysis, the flow rate of the erythrocyte suspension in the dialyser, the osmolarity of the buffer of hypotonic dialysis, the dialysis temperature and resealing temperature and the trans-membranous pressure in the dialyser. The osmotic fragility of the erythrocytes varies from one blood sample to the next and could be a leading biological factor. Thus, L. Boucher et al., Biotechnol. Appl. Biochem. 1996, 24, 73-78, studied the influence of the variations in the osmotic fragility of various populations of red corpuscles on the distribution and final concentration of inositol hexaphosphate. In conclusion, the authors indicate that the initial osmotic fragility of the red corpuscles has a predominant role in terms of the degree of lysis and the variations in internalisation of the active ingredient, and that that osmotic fragility depends on a number of factors, such as the permeability of the red corpuscles, the relationship between surface-area/volume and ion content, the physiological state and age of the donor (see also A. A. Hussain et al., Br. J. Haematol. 1984, 57(4): 716-718), the length of time the blood is stored, the presence of medicaments, illnesses (see also K. Kolanjiappan et al., Clin. Chim. Acta 2002, 326(1-2): 143-149) and treatments. The results obtained, with the flow rate of the erythrocyte suspension being caused to vary, demonstrated the extreme sensitivity of the operating conditions, with a flow rate of from 12 to 14 ml/min depending on whether the red corpuscles belong to a group having a low level of fragility or a group having a high level of fragility.
Therefore, those texts provide initial information on the various factors which influence the osmotic fragility of the red corpuscles and the effectiveness of incorporation by means of the lysis/resealing technique. They allow an understanding of the difficulties encountered in practice, which explain that that technique cannot be applied routinely in human health care.
A quite recent publication summarises the current situation very well. C. G. Millan et al. published, in Journal of Controlled Release 2004, 95: 27-49, a general review of the use of erythrocytes as pharmaceutical vehicles, in which they conclude that, in spite of the interest which they are exciting in human medicine, their development is still very limited today because of the difficulties of storage, risks of contamination and absence of a proven industrial procedure allowing the preparation thereof.
Asparaginase is an enzyme produced from bacterial microorganisms (E. Coli or Erwinia) which hydrolyses and depletes asparagine, an amino acid which is indispensable for synthesising the proteins necessary for cell life, in particular fibroblasts. Some cancerous lymphoblastic cells do not have, unlike normal cells, the capacity to synthesise their asparagine themselves and are dependent on extracellular sources. Treatment by asparaginase therefore deprives them of that constituent, leading to their death. This antimitotic is selective with respect to tumour cells.
In humans, however, native asparaginase induces the production of antibodies which are present in more than 70% of patients on average, leading to an increase in the clearance of asparaginase and allergic reactions which are sometimes very severe (B. Wang et al., Leukaemia 2003 17, 8: 1583-1588). Thus, although asparaginase is very effective in the treatment of acute lymphoblastic leukaemias, it is highly toxic and may lead to hypersensitivity reactions, ranging from a simple reaction of the urticary type to a full-blown anaphylactic shock. Furthermore, there are observed detrimental effects of the neurological type (disturbances to consciousness), haemostatic type (hypofibrinogenaemia, reduction in the serum level of antithrombin III and other coagulation factors, leading to haemorrhagic and/or thrombotic complications), gastro-intestinal type and pancreatic type (including acute inflammations of the pancreas).
The encapsulation of asparaginase in erythrocytes allows the therapeutic index to be improved (D. Schrijvers et al., Clin. Pharmacokinet. 2003, 42 (9): 779-791). Therefore, it would be extremely advantageous to provide a process which allows asparaginase to be encapsulated in erythrocytes in a reproducible and industrial manner.
Furthermore, inositol hexaphosphate has been proposed as a substitute for 2,3-DPG (2,3-diphosphoglycerate) in erythrocytes in order to significantly reduce the affinity of oxygen for haemoglobin and to increase the release of oxygen in tissues (EP-A-0 101 341). U.S. Pat. No. 4,321,259, U.S. Pat. No. 5,612,207 and U.S. Pat. No. 6,610,702 describe the incorporation of that substitute in erythrocytes and the use thereof in various therapeutic applications. They include an indication as an additive for a cancer treatment by means of radiotherapy, in order to improve the oxygenation of hypoxic tumours and their sensitivity to radiotherapy. However, that indication is not accompanied by any feasibility element.
For encapsulation, U.S. Pat. No. 4,321,259 uses the fusion between erythrocytes and liposomes which contain inositol hexaphosphate. U.S. Pat. No. 5,612,207 uses a technique by electroporation. U.S. Pat. No. 6,610,702 sets out an improvement in the electroporation technique, by inositol hexaphosphate being associated with ammonium cations in order to form a biocompatible hydrosoluble complex which can promote introduction in erythrocytes. Finally, in Biotechnol. Appl. Biochem. 1996, 24, 73-78, described above, L. Boucher et al. study the introduction of inositol hexaphosphate in erythrocytes by the lysis/resealing technique. Therefore, various routes are available to the person skilled in the art in order to introduce that compound into erythrocytes. As C. G. Millan et al. (above), mentions, when making reference to inositol hexaphosphate for the transport of oxygen in general, however, the use of erythrocytes incorporating a molecule such as inositol hexaphosphate nowadays encounters the absence of a proven industrial procedure.